The Mechanistic Conception of Life

This behavior of the eggs under the influence of foreign sperm is comprehensible under the assumption that the spermatozoon also causes the development of the egg through two agencies; one of these agencies is a cytolytic substance, a so-called lysin. This substance is probably situated at the surface of the spermatozoon. This lysin only calls forth the membrane formation and it acts like the but>Tic acid in the method of artificial parthenogenesis. The second agency seems to be more in the interior of the spermatozoon and it exercises an influence similar to the short treatment of the egg with a hypertonic solution. A normal development will result only if the spermatozoon enters the egg since in this case only both agencies, the cytolytic and the corrective, get into the egg. We have already mentioned that foreign spermatozoa penetrate only slowly into the egg. If a spermatozoon penetrates partially through the surface of the egg without entirely penetrating into the protoplasm, enough of the lysin sticking to the surface of the spermatozoon can be dissolved to cause the cytolysis of the surface film of the egg which gives rise to the membrane formation. Such eggs receive from the spermatozoon only the lysin, and they act therefore as if only the membrane formation had been called forth in them by the treatment with but>Tic acid, since in the formation of the membrane the spermatozoon is thro\\'n out.

In the eggs of Strongylocentrotus purpuratus the membrane formation can in general only be called forth by living star-fish sperm while the extract of dead star-fish sperm in the same concentration remains without effect. This fact is of importance to disprove the possibilit^'that the membrane formation in these experiments was caused by star-fish blood which was added with the sperm. That it is possible to separate a lysin from the sperm can be proved for the eggs of another species of sea-urchins, namely, Strongylocentrotus franciscanus, the eggs of which are very sensitive to lysins. In these eggs it is possible to call forth the membrane formation with a very dilute watery extract of the sperm of star-fish which was killed by heating it to 60° C, or more. Such eggs can be caused to develop into plutei by treating them after the membrane formation for a short time with hypertonic sea-water. In the place of star-fish sperm the sperm of other foreign species can be used. I have called forth the membrane formation in the sea-urchin egg with the living sperm of sharks or even roosters. Such eggs act as if only the membrane formation with the aid of butyric acid had been caused. At room temperature they begin to develop but they are sickly and soon perish. If, however, they are treated afterward with a hypertonic solution they develop into normal plutei. In this case only the lysin entered the egg but not the spermatozoon. It was, therefore, necessary to treat such eggs subsequently with hypertonic sea-water in order to cause them to undergo normal development at room temperature.

The idea that a lysin contained in the spermatozoon is the formative stimulus which causes the egg to develop can be tested experimentally. We know that blood contains lysins which destroy the blood corpuscles of foreign species, while it does not destroy the cells of the same species. If the idea is correct that the spermatozoon acts upon the egg through a lysin which calls forth the membrane formation it should be possible to call forth the membrane formation in the unfertilized egg of the sea-urchin by foreign blood and such is the case. I was able to show three years ago that the blood of certain

worms, namely SipuncuUdes , can call forth the membrane formation in the sea-urchin egg even if it is diluted a hundred times or more with sea-water. This effect is not produced in the eggs of every female sea-urchin but of only about 20 per cent of the females. I think the difference is caused by differences in the permeability of the eggs for lysins; and the degree of permeability seems to vary slightly for the eggs of different females. Instead of wasting time on an examination of the effects of the blood of invertebrates^ I examined the effects of the blood serum of warm-blooded animals. I succeeded in causing membrane formation in the sea-urchin egg (purpuratus) with the blood serum of cattle, sheep, pigs, and rabbits; and such eggs behaved like the eggs which had been treated with the living sperm of roosters or with butyric acid. They began to develop, but they became sickly at room temperature and soon disintegrated. If, however, they were treated after the membrane formation for a short time with a hypertonic solution they developed at room temperature. The blood, therefore, contains the lysin, but not the second substance necessary for the full development. It is, therefore, necessary to substitute for the action of the latter the treatment with a hypertonic solution if we wish to call forth a normal development of the egg treated with serum.

The lysin of the blood is like that of the spermatozoon relatively resistant to heat. The blood does not lose its power to call forth membrane formation by heating it for some time to 60° or 65° C.^ It is curious that SrCl, and Babl2 increase the membrane-forming power of the blood. Not only blood but also the extracts of the organs of foreign 1 Since this was written the blood and the extracts of organs of a number of invertebrates were used successfully to produce the membrane formation and development of the egg of the sea-urchin.

2 The substance which causes membrane formation can be precipitated with acetone (Loeb, P fingers Archit, CXXIV, 37, 1908). species call forth membrane formation in the sea-urchin egg. An extract of the coecum of the star-fish was very effective. We have already mentioned the fact that the extract of dead sperm of foreign species, e.g., of star-fish, certain mollusks, certain worms, sharks, fowl, causes membrane formation in the eggs of franciscanus. Experiments with the extract of dead sperm of their owti species on the egg oi franciscanus or purpuratus fail; and the same is true for extracts from the tissues of these species.^ What causes this difference in the action of the lysins from their own and a foreign species? We know that the lysins of our own blood do not hurt our cells while they hurt the cells of foreign species. There exists, therefore, an immunity of the eggs as well as of the rest of the cells against the lysins of their own blood or tissues.

Our experiments throw a light upon the nature of this immunity. If the lysins contained in our blood do not injure our cells it can only be due to one of two facts: The lysins of our own blood can either not diffuse into our cells, while they can diffuse into the cells of foreign forms, or the cells contain antibodies against the lysins of their own body, but not against those of foreign species. As far as the lysins of the blood are concerned we cannot decide between the two possibilities. We can, however, reach a decision for the lysins of the spermatozoa. The extract from the dead sperm of the sea-urchin is ineffective for the eggs of the sea-urchin solely for the reason that it cannot diffuse into the sea-urchin egg. For if the seaurchin lysin is carried by the living sea-urchin spermatozoon (which acts as a motor for the lysin) into the sea-urchin egg, the lysin is very active and probably more active than the lysin of foreign species. If the sea-urchin egg contained an antibody against the lysin of the sea-urchin sperm, the seaurchin sperm should not be able to call forth membrane formation when it enters the sea-urchin egg.

1 If eggs were sensitized with SrCl., they could be caused to develop by extracts from the coecvun of the sea-urchin, though this was trueonljexceptionally. We now understand the paradoxical fact, that by foreign sperm we can cause membrane formation and development of the sea-urchin egg in two different ways: namely, first by the living sperm and second by the extract from the dead sperm; while the sperm of the same species can only cause the eggs to develop when it is alive. We now understand the fact alluded to at the beginning of this chapter that my first experiments to cause the development of the egg with extract of sperm did not succeed, since I took it for granted that it was necessary to use the extract of the sperm of the same species from which the eggs were taken. The lysins in this case were not able to diffuse into the egg.

The further unraveling of the nature of the immunity of the egg-cell against the dissolved lysins of the blood and the tissues of the same species depends upon the explanation of the fact that the lysins of a species cannot diffuse into the egg of the same species. It would be of interest if the same principle formed for the immunity of the egg-cell would hold also for the immunity of the body-cells against the lysins in the blood of their o%vn species.

We may, therefore, say that the substance to which the sperm owes its fertilizing power is a lysin and we may express the suspicion that the lysins which we have thus far kno\\TL only as protective agencies against bacteria play a great physiological role in the mechanism of life phenomena. We may call our theory of the developmental action of the spermatozoon the lysin theory; thereby designating that the impulse for the development of the egg is given by a lysin contained in the spermatozoon. In artificial parthenogenesis we substitute for the natural lysin a cytolytic substance. Aside from the lysin action the normal development demands, as a rule (but not always), a second corrective influence which in artificial parthenogenesis may be given by a hypertonic solution.

The experiments on the artificial parthenogenesis of other forms of animals show that the eggs of different animals possess a varying tendency for parthenogenetic development. There are eggs which can easily be induced to develop, so easily in fact, that the experimenter cannot always be sure whether he has caused the development by a substance applied by him or whether some accidental condition of the experiment was responsible. The eggs of the silkworm, of the star-fish, and of certain worms belong to this class. In working with starfish eggs we can observe that occasionally a few of them develop in normal sea-water, apparently without any demonstrable cause, into swimming larvae. The eggs of the Californian sea-urchin Strongylocentrotus purpuratus, on the other hand, show not the slightest tendency to segment parthenogenetically ; only the above-mentioned very specific and quantitative method causes them to develop. For this reason I selected these eggs for the investigation of the nature of the experimental stimulus, since I could always be sure that the same stimulus gave the same results; while, e.g., in the star-fish eggs we can never be perfectly certain that some internal condition in the egg or some overlooked unimportant secondary condition in the experiment may not have caused the development. Although eggs with such a strong tendency for spontaneous development as the star-fish eggs are not the best material for the study of the nature of the developmental stimulus yet we have to answer the question how it happens that some eggs have a greater tendency for parthenogenetic development than others.

Mathews has observed that by gently shaking the star-fish eggs the number of eggs which develop "spontaneously" can be increased. I made a similar observation in the eggs of Amphitrite, an annelid. In the eggs of the sea-urchin nobody has ever been able to obtain such a result. I am inclined to believe that if a sea-urchin should be found, the eggs of which possess a greater tendency to develop spontaneously, it might also be found that the number of eggs developing spontaneously might be increased by agitation.

I tried whether it is possible to cause the eggs to cytolyze also mechanically. If we exercise only a slight pressure with a finger upon the ovary of a star-fish we find that many of the eggs which afterward leave the ovary are cytolyzed. This cytolysis is not caused by a bursting of the egg membrane; on the contrary, in this case the cytolysis of the egg is, as usual, preceded by the formation of a membrane of fertilization and this membrane remains intact in the star-fish egg which is caused to cytolyze by mechanical pressure. In the sea-urchin egg, however, it is impossible to produce cytolysis by a slight pressure.

The eggs of the star-fish which develop spontaneously first form a membrane. Shaking causes a development of the starfish eggs only if the-shaking first leads to a membrane formation. The greater tendency of the star-fish to develop spontaneously is, therefore, due to the greater ease with which cytolysis can be produced in this egg. How can mere agitation or pressure call forth membrane formation or cytolysis ? It seems to me that this fact is most easily understood under the assumption, first suggested by Biitschli, that the cytoplasm is an emulsion. It would then follow that the membrane formation as well as the cytolysis depends upon the destruction of this emulsion. We know that different emulsions have a different degree of durability. The eggs which upon gentle pressure undergo cytolysis have an emulsion with a lesser degree of durability than the eggs in which pressure has no such effect. Let us assume that membrane formation as well as cytolysis depends upon the destruction of an emulsion; in this case the membrane formation depends upon the destruction of the emulsion in the cortical layer of the egg only. The lysin of the egg destroys only the emulsion in

the cortical layer of the egg and thereby causes development. The greater tendency of the eggs of certain animals for spontaneous parthenogenetic development thus depends upon the relatively small degree of durability of the emulsion which constitutes the cortical layer of the egg. But it should be stated that this hypothesis is not essential for the lysin theory of the activation of the egg. The assumption that the membrane formation is only a superficial cytolysis of the egg presupposes that the cortical layer of the egg is different from the rest of the cytoplasm. Biitschli had already reached such a conclusion on the basis of histological observations. I am inclined to accept this view on the basis of my observations on the action of cytolytic agencies on the unfertilized egg. The action of these agencies on the unfertilized egg always occurs in two stages which are often separated from each other by a considerable interval of time. The first stage is the cytolysis of the superficial layer; the second stage is the cytolysis of the rest of the egg. This is most obvious in experiments with weak solutions of saponin or solanin in sea-water. In this case first a membrane formation occurs, then a pause ensues, often of several minutes, and then cytolysis of the whole egg follows. If instead of saponin benzol is used a pause can also be observed between membrane formation and cytolysis of the whole egg but this pause is short, often only a fraction of a second, or at the best a few seconds.

It can also be shown directly that there is a qualitative difference between the cortical layer of the protoplasm and the rest. If for the artificial membrane formation the lower fatty acids, from the formic to the capronic acid, are used, cytolysis of the cortical layer only is observed, i.e., membrane formation follows but no cytolysis of the whole egg. If, however, the higher fatty acids of the same series from the heptylic acid on and upward are applied the membrane formation is always followed after a short pause by a cytolysis of the whole egg.

The lysins contained in the blood and the spermatozoon act according to my present experience only upon the cortical layer of the cytoplasm but not on the rest of the egg. We get a membrane formation and development but not a cytolj'sis of the whole egg. If we go back to the idea of Butschli that protoplasm has the structure of an emulsion we are led to the view that the emulsion of the cortical layer of the egg differs from that of the rest of the egg. There are certain cytolytic agencies which destroy only the cortical layer; while all general cytolytic agencies destroy the cortical layer as well as the rest of the egg.

How can the cytolysis of the cortical layer of the egg lead to a membrane formation? Von Knaffl has expressed the following view on this point: ''Protoplasm is rich in lipoids, it is probably mainly an emulsion of these and of proteins. Every physical and chemical agency which is able to liquefy lipoids calls forth a cytolysis of the egg. The protein of the egg can only swell or be dissolved if the state of the lipoids is altered by chemical or physical means. The mechanism of cytolysis consists in the liquefaction of the lipoids and the subsequent swelling or liquefaction of proteins by absorption

We can accept this with a slight modification which refers to the nature of the emulsion. An emulsion requires not only two substances or phases as von Knaffl assumes but in addition a third substance. The third substance serves the purpose of making the emulsion more durable (Lord Rayleigh's theory). The droplets of the emulsion are surrounded by a thin layer of a substance which lessens the surface tension between the droplet and the second phase of the emulsion. I assume that only this stabilizing substance consists of lipoids, especially cholesterin. The two other phases which constitute the emulsion need not be lipoids. To fix our ideas provisionally we may assume that these phases are first protein with little water and second water with little protein. The existence of these two phases has been established by Hardy. The emulsion at the surface of the egg consists, according to this view, of a system of protein droplets poor in water surrounded by a stabilizing film of a lipoid (cholesterin or lecithin). If the seaurchin egg is treated with a lipoid solvent like benzol the stabilizing film of cholesterin is dissolved and the protein droplet can absorb water. If we use saponin the film is destroyed by the precipitation of cholesterin by saponin. The absorption of water leads to the lifting up of the surface film which surrounds the egg.^

We wish to add a few remarks concerning the nature of this surface film, although this does not belong to our problem. According to Overton and Koeppe the surface film of cells consists of lipoids, and according to Koeppe cytolysis is determined by the solution or tearing of this film. This view is not tenable, since the surface film which is lifted off in the form of the fertilization membrane does not consist of a lipoid but of protein. This is suggested by the fact that this membrane is absolutely insoluble in any lipoid solvent. Moreover, this membrane remains perfectly intact when the egg is transformed

Since we can cause the formation of a membrane of fertilization in the star-fish egg by gentle agitation or mere pressure, 1 We have assumed here that the fertilization membrane is preformed in the imfertilized egg and lifted up in consequence of the cytolysis of the layer beneath it. As I stated in my book on Die chemische Entwicklungserregung des tierischen Eies, it is also possible that the fertilization membrane is a membrane of precipitation formed through the reaction of a constituent of the liquefied cortical layer with a constituent of the sea-water (Ca?). It is immaterial for the problem discussed in this paper which view we adopt temporarily.

this membrane is apparently preformed in the unfertilized egg; and if this be true the process of membrane formation must consist in the lifting up of a preformed film from the underlying cytoplasm through the entrance of sea-water between this film and the cytoplasm. In this process the surface film undergoes a change, since the spermatozoon can enter into the egg before but not after the membrane formation. That merely a change in the nature of the surface film prevents the entrance of a spermatozoon into the egg after the membrane formation can be proved by the fact that if we tear the membrane mechanically a spermatozoon can penetrate into the egg. This proves that the surface film, even if it is already preformed in the unfertilized egg, has different qualities or a different structure when it is in close contact with the cytoplasm than when it is lifted off from the cytoplasm by a layer of sea-water.

We have assumed that the membrane formation is determined by the action of a lysin or cytolytic agency upon the cortical layer of the egg, whereby a protein in this layer absorbs sea-water, and is thereby dissolved. This assumption leads to two consequences: first it must be possible to show that the fertilization membrane is permeable for sea-water and crystalloid substances but impermeable for colloids. The correctness of this view can be proved. If we add to the sea-water, containing eggs with a fertilization membrane, a certain quantity of dissolved white of egg, tannin, or blood serum, the membrane collapses and closes tight around the cytoplasm. The reason is that almost all the liquid which existed between the membrane and the cytoplasm diffused into the surrounding seawater. If the eggs are brought back into normal sea-water (free from protein) it diffuses again into the space between the membrane and the cytoplasm, and the fertilization membrane resumes its former distance from the cji^oplasm and its round shape. The membrane is, therefore, impermeable for the colloids dissolved in sea-water.

If salts are added to the sea-water or if it is diluted by the addition of distilled water the tension and the diameter of the membrane do not change. This proves that the membrane is permeable for salts, but not for colloids, and that the lifting up of the fertilization membrane is determined by the swelling and subsequent liquefaction of a colloid. This dissolved colloid exercises an osmotic or colloidal pressure and sea-water must diffuse from the outside imder the fertilization membrane of the egg until the tension of this membrane equals the osmotic or colloidal pressure of the dissolved colloid. This explains also why it is that the fertilization membrane as a rule assumes a spherical shape.

We now can understand why not in all cases of fertilization a distinct fertilization membrane is formed. This may be due to the fact that the degree of swelling of the colloid of the cortical layer varies under different conditions. We now possess a pretty complete picture of what happens to the egg in the case of ''formative stimulation," i.e., when it is caused to develop. Through a lysin or some other cytolytic agency a certain substance of the cortical layer, presumably a lipoid, is dissolved or precipitated, whereby a protein substance of that layer is able to absorb water and swell. Formerly it was thought that the spermatozoon caused the development of the egg by carrying a ferment or enzyme into it and that this ferment set the mechanism of development into action. Others expressed the opinion that the entrance of the sperm nucleus or of a centrosome was responsible for the development. We see, however, that it suffices to call forth the artificial membrane formation in the unfertilized sea-urchin egg, in order to observe after two or three hours the formation of normal astrospheres or spindles. This disproves the suggestion that the fusion of egg and sperm nucleus is essential for the development of the egg.^

1 The fusion of the nuclei is of course of importance for the transmission of paternal qualities. The ferment theory of the activation of the egg by the spermatozoon is also wrong. If it were correct the velocity of development should be accelerated if not doubled if two spermatozoa enter the egg instead of one; or if fertilization by sperm and artificial parthenogenesis are superposed in the same egg. But this is not the case. In neither case is a shortening of the time which elapses between two successive periods of segmentation observed.^

The further development will be connected with the question how can the cytolysis of the cortical layer of the egg lead to its development? I may mention the possibility that the cytolysis of the cortical layer facilitates the diffusion of oxygen or of HO ions (bases) or other substances, necessary for the development, into the egg. Let us summarize our results concerning the activation or formative stimulation of the egg. For the normal development at least two agencies are required: the one is the cytolysis of the thin cortical layer of the egg. Any agency which causes this cytolysis (without causing the cytolysis of the rest of the egg) induces development. The spermatozoon as well as the blood and the tissues contain a substance (lysin) which causes only cytolysis of the cortical layer. The lower fatty acids, from formic to capronic, cause only the cytolysis of the cortical layer. Since most cytolytic agencies cause a cytolysis of the whole egg they can be used only if the eggs are withdrawn from their influence after the cortical layer is destroyed but before the rest of the egg has undergone destruction.

Cytolysis of the cortical layer leads often but not always to the formation of the membrane of fertilization. Since all cytolytic substances are lipoid soluble (or destroy lipoids) it is probable, but not proved, that the formative 1 Another reason is that the velocity of segmentation is purely determined by the egg, no matter what is the nature of the spermatozoon. stimulus in the activation of the egg consists in a liquefaction or precipitation or some other modification of the lipoids of the cortical layer of the egg which results in an imbibition or solution of a colloidal substance of the cortical layer. If the cytoplasm has the structure of an emulsion it is possible that lipoids form the stabilizing envelope for the droplets which, according to Lord Rayleigh, is necessary for the durability of the emulsion. The cytolysis of the cortical layer of the egg causes its development, but this development is often abnormal and comes prematurely to a standstill. In order to induce a more normal type of development a second agency is often required, the mode of action of which is not yet so clearly understood as that of the cytolytic agency, namely, a short treatment of the egg with a hypertonic solution containing oxygen or a longer inhibition of the development of the egg in normal sea-water which is free from oxygen. The spermatozoon carries in addition to the lysin a second substance into the egg, which acts similarly to the hypertonic solution in our method of artificial parthenogenesis .

The unfertilized egg dies in a comparatively short time, while the act of fertilization gives rise to a series of generations which, theoretically at least, is of infinite duration. The act of fertilization is, therefore, a life-saving act for the egg. The question arises, in which way can the spermatozoon save the life of the egg ? If the ovaries of a star-fish are put into sea-water the eggs are shed. They are generally immature, and in this condition they cannot be fertilized, either by spermatozoa or by chemical means. If they remain, however, for some time in sea-water, all or a number of them gradually become mature; that is to say, their nuclear mass is diminished by the extrusion of the two so-called polar bodies. If immediately after the extrusion of the polar bodies sperm is added, the eggs develop. They can at that period likewise be caused to develop by certain chemical and physical agencies.

Ten years ago I made the following observations. If the eggs are not caused to develop by sperm or by physico-chemical agencies, they perish very rapidly. At summer temperature they may die in from four to six hours. The death of the egg manifests itself morphologically in a darkening and blackening of the otherwise clear egg. I found that the death of the egg can be prevented by withdrawing the oxygen, or by diminishing the rate of oxidations in the egg through the addition of a trace of potassium cyanide. The life-saving action of lack of